Isolation And Identification Of Avian Infectious Bronchitis Virus And Establishment Of A Multiplex PCR Method For The Diagnosis Of Five Chicken Viruses

Infectious bronchitis(IB),Avian influenza(AI),Newcastle disease(ND),Infectious bursal disease(IBD)and Fowl pox(FP)are important Infectious diseases in poultry.Due to the large antigenicity difference of IBV and the wide application of vaccines targeting different serotypes of IBV in China,it is very important to isolate the local endemic strains and analyze the genetic evolution of their S1 gene for the research of sustainable vaccination strategies.IB,AI,ND,IBD and FP are highly infectious,often with mixed infection or secondary infection.To establish a simple and rapid multiplex PCR diagnosis method that can simultaneously detect five pathogens is helpful for the prevention and rapid diagnosis of poultry diseases1.Tissue samples of suspected IB chickens were collected aseptically from a chicken farm in Urumqi City for virus isolation and identification.By molecular biology experiment,blood clotting,chicken embryos small test,the new town of interference test,determination of chicken embryos half the amount of infection,pathogenic test and some physical and chemical properties test showed that isolates in chicken embryos even five generations appear dwarf embryo phenomenon and can inhibit the NDV in chicken embryo proliferation,only after 1%of pancreatic enzyme treatment can to chicken red blood cell aggregation effect,by calculating the EID5010^-4.58/0.1m L,for moderate virulence strains,poison attack group than normal chick tracheal there were a lot of mucus and intumescent,lung performance for breathing type strain characteristics,is sensitive to heat,grease solvent,Some tolerance to 1%trypsin,less tolerance to acids than to bases.The disease was confirmed to be the original IBV strain.2.The sequencing results of S1 gene of the isolates were compared with the published reference S1 gene of Gen Bank.The results showed that the isolates were most closely related to IBV/I0221/17/China strain,and formed a separate branch.It formed an independent evolutionary group with TC07-2,GX-NN09032,CK/CH/IBTZ and CK/CH/GD/HY16 strains,and all belonged to TC07-2/GVI-1 IBV.The homology with the commonly used domestic vaccine strains such as H120,H52 and M41 is only 59?65%,and its S protein cleavage site sequence is HRRKR,which is named as IBV/CK/CH/01/2020 strain3.According to the IBV,AIV,NDV,IBDV and FPV gene sequences in the NCBI nucleic acid database,design 5 pairs of specific primers,optimize annealing temperature,reaction cycle number and primer concentration,conduct specificity and sensitivity tests,and conduct clinical testing.The results showed that the established multiplex PCR method can effectively amplify genomic DNA or c DNA of IBV,AIV,NDV,IBDV and FPV,and can effectively amplify goose parvovirus(GPV),avian infectious laryngotracheitis virus(ILTV),and avian type 4.The nucleic acid of paramyxovirus(APMV-4)has no specific amplification and has strong specificity.The minimum detection limits for recombinant plasmids of IBV,AIV,NDV,IBDV and FPV are 1×10⁵ copies/?L and 1×10⁴,respectively.Copy/?L,1×10⁴ copy/?L,1×10⁴ copy/?L,1×10⁴copy/?L,the coincidence rate of 41 naturally infected poultry samples with single PCR detection was 90.32%,indicating the established multiple The PCR detection method has good specificity,sensitivity and clinical applicability.This study mainly studied the variation of the endemic strains in a chicken farm in Urumqi and provided experimental data for elucidating the genetic variation of IBV.It provides a simple and rapid multiplex PCR method for the identification of single or mixed infection of five chicken virus diseases,and provides technical support for the effective prevention,control and extermation of these avian virus diseases.